CN104198634A - Method for establishing Sishen tablet fingerprint spectrum - Google Patents
Method for establishing Sishen tablet fingerprint spectrum Download PDFInfo
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- CN104198634A CN104198634A CN201410389089.5A CN201410389089A CN104198634A CN 104198634 A CN104198634 A CN 104198634A CN 201410389089 A CN201410389089 A CN 201410389089A CN 104198634 A CN104198634 A CN 104198634A
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Abstract
The invention discloses a method for establishing a Sishen tablet preparation fingerprint spectrum. The method comprises the following steps: preparing a reference solution which contains 10-100mg of psoralen, isopsoralen and schisandrin per milliliter; and removing coatings from Sishen tablets, weighing out 0.1-10g of Sishen tablets without coating, performing ultrasonic extraction with methanol, performing filtration to obtain a test solution, precisely sucking the test solution, injecting the test solution into a liquid chromatograph, and determining in accordance with a liquid chromatography, thereby obtaining the fingerprint spectrum. The method is performed under the following chromatographic conditions: octadecyl silane-bonded silica gel serves as stuff of a chromatographic column, a mobile phase refers to a gradient eluent consisting of acetonitrile and water, the detection wavelength is 220-360nm, the column temperature is 20-50 DEG C, the flow velocity is 0.5-1.5ml/min and the time is 30-90 min. The method is simple and stable, has high precision and good reproducibility, and is capable of rapidly and correctly distinguishing the authenticity and the quality of products.
Description
Technical field
The present invention relates to the method for quality control of four refreshing sheets, be specifically related to construction method and the finger-print of four refreshing sheet high-efficiency liquid-phase fingerprints, belong to traditional Chinese medicine and pharmacy technical field.
Background technology
Traditional Chinese medicine fingerprint refers to utilization modern analytical technique, and Chinese medicine chemical information is characterized and described in the mode of figure, is common in certain or certain several Chinese crude drug, has the chromatogram of distinctive a few constituents or the collection of illustrative plates of spectrum.Current, traditional Chinese medicine fingerprint Quality Control Technology, as the detection means of controlling traditional Chinese medicine quality stability and homogeneity, is one of gordian technique of the modernization of Chinese medicine, and this is significant to effectively controlling the quality of Chinese medicine.Chromatographic fingerprinting Quality Control Technology is the bright spot of current traditional Chinese medicine quality research.Chinese medicine for complicated component, that single active ingredient of past is incomplete as the way of quality control index, chromatographic fingerprinting control model be take stratographic analysis as means, therefrom obtain the information of reaction Chinese medicine inherent quality, by the analysis comparison to fingerprint characteristic, authenticity, consistance and the stability of evaluating Chinese medicine inherent quality, this is the best techniques of present stage comprehensive evaluation traditional Chinese medicine quality.It is reported, the main manufacturing enterprise of Japanese Kampo medicine just adopts liquid-phase fingerprint to control quality in enterprises the eighties in 20th century, and the countries such as the U.S., Germany, France also adopt finger-print Quality Control Technology to plant herbal medicine.Along with applying of traditional Chinese medicine, it is found that, as the time product of theory of traditional Chinese medical science, Chinese medicine is herbal mixture especially, and wherein any contained composition all can not represent its whole curative effect.People recognize gradually, the quality of the reaction Chinese medicine inherence that the existing quality standard with reference to Western medicine quality control model can not be appropriate, and finger-print is as Chinese herbal medicine and extraction of substance amount control method thereof, and the international institute of Yi Wei knows together at present.
Four refreshing sheets are comprised of Six-element medicines such as nutmeg (system), evodia rutaecarpa, Psoralea corylifolia, the fruit of Chinese magnoliavine, rhizoma zingiberis, dates, have the kidney of helping loose cold, the effect of antidiarrheal dissipate-swelling.Clinical be used for the treatment of to suffer from a deficiency of the kidney catch cold, borborygmus tripe is swollen, half congealed rushing down just before dawn, food is not changed, endless diarrhea, a little less than face corpus luteum.
At present, country and enterprise are to adopt thin-layered chromatography to differentiate the schizandrin in evodia rutaecarpa, the fruit of Chinese magnoliavine and the psoralen in Psoralea corylifolia, Isopsoralen to the quality control of four refreshing sheets.Slander intelligent, Fang Xiaoming etc., " CHINA JOURNAL OF CHINESE MATERIA MEDICA " 2006, the 6 phases adopt HPLC method measure four refreshing sheets in the content of psoralen and isopsorapen; Celebrating is pretty to be waited in " Journal of Jilin Medical College " 2008,29(6) the middle content that adopts RP-HPLC method to measure psoralen and isopsorapen in four refreshing sheets.
Below are all discriminating or assays that indivedual compositions are carried out, cannot characterize the chemical feature of four refreshing sheets comprehensively, finger-print, as the method for quality control of Chinese herbal medicine and prescribed preparation, can more effectively characterize the quality of Chinese medicine on the whole.At present, the fingerprint spectrum method of relevant four refreshing sheets there is not yet report.
Summary of the invention
Object of the present invention, is to provide a kind of construction method of four refreshing tablet preparation finger-prints.Be the quality control of four refreshing sheets and the true and false and differentiate a kind of new method is provided, and method obtains the standard finger-print of four refreshing sheets thus.
The fingerprint spectrum method that the present invention sets up has good precision, repeatability and stability, can be used in the quality control of four refreshing sheets.By the method, can guarantee quality stability, consistance and the controllability of four refreshing sheets, thereby guarantee security and the validity of four refreshing sheets.
Four refreshing sheets described in the present invention are by nutmeg 189g, evodia rutaecarpa 94g, and Psoralea corylifolia 377g, fruit of Chinese magnoliavine 189g, rhizoma zingiberis 94g, date 189g is prepared from.Concrete preparation method is: above Six-element, and date boiling secondary adds 8 times of amounts of water at every turn, decocts 1 hour, collecting decoction, reduced pressure concentration becomes thick paste; Nutmeg, evodia rutaecarpa, Psoralea corylifolia, Fructus Schisandrae Chinensis powder are broken into meal, with 60% ethanol, make solvent, carry out diacolation.Rhizoma zingiberis pulverizing medicinal materials becomes meal, with ethanol, makes solvent, carries out diacolation.Merge ethanol percolation liquid, reclaim ethanol, reduced pressure concentration becomes thick paste.Water, alcohol thick paste merge, and add right amount of auxiliary materials, mix, and drying under reduced pressure, pulverizes, and adds appropriate amount of auxiliary materials, according to conventional method, makes corresponding four refreshing tablet preparations.
Therefore first object of the present invention is to provide a kind of method for building up of four refreshing sheet HPLC finger-prints, said method comprising the steps of:
(1) preparation of reference substance solution: it is appropriate that precision takes psoralen, Isopsoralen, schizandrin reference substance respectively, with methyl alcohol, be mixed with every ml containing the solution of psoralen, Isopsoralen and schizandrin 10-100 μ g, obtain reference substance solvent, standby.
(2) preparation of need testing solution: after four refreshing sheets remove dressing, precision takes 0.1～10g, with the ultrasonic extraction 10～60min of methyl alcohol, extract filters with miillpore filter, obtains need testing solution;
(3) adopt liquid chromatography for measuring finger-print: the accurate need testing solution of drawing injects high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains finger-print.
(4) adopt the chromatographic condition of liquid chromatography for measuring finger-print: chromatographic column be take octadecylsilane chemically bonded silica as filler; The gradient eluent that mobile phase is comprised of mobile phase A and Mobile phase B, mobile phase A is acetonitrile, Mobile phase B is water; Detect wavelength 220nm～360nm; 20～50 ℃ of column temperatures; Flow velocity 0.5～1.5mL/min; Time 30～90min.
The preparation of above-mentioned steps (2) need testing solution is: after four refreshing sheets remove dressing, precision takes 0.1-10g, puts in tool plug conical flask, and precision adds methyl alcohol 10-100ml, close plug, weighed weight, soaks, after ultrasonic processing, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate, obtain need testing solution.
Above-mentioned steps (3) is measured finger-print: accurate need testing solution 5～25 μ l that draw inject high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtain finger-print.
The described chromatographic condition of above-mentioned steps (4) is: mobile phase is the gradient eluent that water and acetonitrile form, water 95-20%: acetonitrile 5-80%, and 35 ℃ of column temperatures, detection wavelength is 246nm, and flow velocity is 1.0ml/min, and be 70min analysis time.
The preparation of the described reference substance solution of above-mentioned steps (1) is: precision takes psoralen, Isopsoralen, schizandrin reference substance in right amount as for volumetric flask, with methyl alcohol, be diluted to scale, be mixed with every 1ml containing the solution of psoralen 30 μ g, Isopsoralen 30 μ g, schizandrin 50 μ g, in contrast product solution;
The preparation of the described need testing solution of above-mentioned steps (2) is: after four refreshing sheets are removed dressing, precision takes 0.5g, puts in tool plug conical flask, and precision adds methyl alcohol 25ml, close plug, weighed weight, soaks, ultrasonic processing 45 minutes (50kHz, room temperature), lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, obtain need testing solution;
The described chromatographic condition of above-mentioned steps (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase is the gradient eluent that acetonitrile and water form; Column temperature: 35 ℃; Detect wavelength 246nm; Flow velocity: 1.0ml/min; Analysis time: 90min
The gradient elution that above-mentioned steps (4) is described, preferred gradient elution program carries out with following volumetric concentration proportioning.Mobile phase A is acetonitrile, and Mobile phase B is water.
The refreshing sheet finger-print of table 1. four preferred gradient elution program
| Time | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 5 | 95 |
| 20 | 30 | 70 |
| 25 | 40 | 60 |
| 60 | 80 | 20 |
| 70 | 80 | 20 |
The described mensuration of above-mentioned steps (3) is that the accurate need testing solution 10 μ l that draw inject high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains four refreshing sheet finger-prints.
Compared with prior art, the present invention has the following advantages;
1. the four refreshing sheet HPLC finger-prints that method provided by the present invention is set up are all greater than 0.9 with the similarity that contrasts collection of illustrative plates, can effectively characterize its quality, are conducive to the quality of overall monitor product;
2. the present invention has set up four refreshing sheet HPLC characteristic fingerprint pattern common patterns, 15 total peaks have been demarcated, the fingerprint pattern technology content of setting up is high, avoided only measuring individualized unicity and the one-sidedness of minute carrying out quality control that studies, the information that has fully reflected on the whole four refreshing sheet chemical compositions, has reduced the possibility of artificial treatment product requisite quality;
3. to have method easy in the present invention, stable, precision is high, high repeatability and other advantages, can differentiate quickly and accurately that the true and false of product is good and bad.
Below by embodiment and accompanying drawing, illustrate in detail technical scheme of the present invention, cited embodiment and accompanying drawing are to the present invention unrestricted.
Accompanying drawing explanation
Fig. 1 is four refreshing sheet reference substance collection of illustrative plates.
Fig. 2 is four refreshing sheet sample finger-prints.
Fig. 3 is 10 batches of four refreshing sheet sample finger-prints.
Fig. 4 is spectrogram after 10 batches of four refreshing sheet sample finger-print couplings.
Fig. 5 is four refreshing tablet preparation standard finger-prints.
Fig. 6 is the finger-print of nutmeg medicinal material.
Fig. 7 is the finger-print of evodia rutaecarpa medicinal material.
Fig. 8 is the finger-print of corylifolia L.
Fig. 9 is the finger-print of schisandra chinensis medicinal material.
Figure 10 is the finger-print of date medicinal material.
Embodiment
embodiment 1:the fingerprint atlas detection method of four refreshing sheets, comprises the following steps:
1. instrument and reagent
1.1 instrument Shimazu high performance liquid chromatographs, LC-20 UV-detector, Labsolution chromatographic work station.
1.2 reagent psoralens, Isopsoralen, schizandrin reference substance; Acetonitrile (chromatographically pure), secondary redistilled water, phosphoric acid (analyzing pure).
2. high performance liquid chromatography
2.1 chromatographic conditions: chromatographic column be take octadecylsilane chemically bonded silica as filler, Phenomenex chromatographic column: C
18(250 * 4.6 mm, 5 μ m); Adopt gradient elution, mobile phase is the gradient eluent that acetonitrile and water form; Column temperature: 35 ℃; Detect wavelength 246nm; Flow velocity: 1.0ml/min; Sample size: 10 μ L.Theoretical cam curve should be not less than 5000 by psoralen technology.
Gradient elution program is as following table, and mobile phase A is acetonitrile, and Mobile phase B is water.
The refreshing sheet finger-print of table 1. four preferred gradient elution program
| Time | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 5 | 95 |
| 20 | 30 | 70 |
| 25 | 40 | 60 |
| 60 | 80 | 20 |
| 70 | 80 | 20 |
The preparation of 2.2 reference substance solution: precision takes psoralen, Isopsoralen, schizandrin, reference substance in right amount as for volumetric flask, with methyl alcohol, be diluted to scale, be mixed with every 1ml containing the solution of psoralen 30 μ g, Isopsoralen 30 μ g, schizandrin 50 μ g, in contrast product solution;
The preparation of 2.3 need testing solutions: after four refreshing sheets are removed dressing, precision takes 0.5g, puts in tool plug conical flask, and precision adds methyl alcohol 25ml, close plug, weighed weight, soaks, ultrasonic processing 45 minutes, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate, obtain need testing solution;
3. measure: the accurate need testing solution 10 μ l that draw inject high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtain four refreshing sheet finger-prints.See Fig. 1, Fig. 2.
embodiment 2detect the finger-print of 10 batch of four refreshing sheet
Get 10 batches of four refreshing sheets, by embodiment 1 condition, detect, obtain the HPLC collection of illustrative plates of 10 batch samples, as shown in Figure 3.By the comparison of 10 batches of HPLC collection of illustrative plates, carry out similarity evaluation, determine that its feature has peak:
In finger-print, there are 15 features to have peak, show the mean value of the retention time of collection of illustrative plates, peak area and account for total area ratio, specific as follows:
No. 1 peak, average retention time RT is 3.21min, and RSD is 0.11%, and peak area is that 129767, RSD is 15.27%, accounts for 3.28% of total peak area.
No. 2 peaks, average retention time RT is 11.090min, and RSD is 4.09%, and peak area is that 229612, RSD is 10.68%, accounts for 5.80% of total peak area.
No. 3 peaks, average retention time RT is 11.553min, and RSD is 3.81%, and peak area is that 131519, RSD is 12.46%, accounts for 3.32% of total peak area.
No. 4 peaks, average retention time RT is 30.305min, and RSD is 0.22%, and peak area is that 895077, RSD is 5.82%, accounts for 22.62% of total peak area.
No. 5 peaks, average retention time RT is 31.075min, and RSD is 0.18%, and peak area is that 129074, RSD is 5.34%, accounts for 3.26% of total peak area.
No. 6 peaks, average retention time RT is 38.518min, and RSD is 0.18%, and peak area is that 129074, RSD is 5.34%, accounts for 3.26% of total peak area.
No. 7 peaks, average retention time RT is 38.934min, and RSD is 0.19%, and peak area is that 106947, RSD is 5.40%, accounts for 2.70% of total peak area.
No. 8 peaks, average retention time RT is 39.612min, and RSD is 0.18%, and peak area is that 57273, RSD is 5.48%, accounts for 1.45% of total peak area.
No. 9 peaks, average retention time RT is 42.359min, and RSD is 0.16%, and peak area is that 25265, RSD is 7.21%, accounts for 3.17% of total peak area.
No. 10 peaks, average retention time RT is 44.085min, and RSD is 0.17%, and peak area is that 89586, RSD is 5.55%, accounts for 2.26% of total peak area.
No. 11 peaks, average retention time RT is 46.327min, and RSD is 0.17%, and peak area is that 50819, RSD is 6.32%, accounts for 1.28% of total peak area.
No. 12 peaks, average retention time RT is 46.884min, and RSD is 0.16%, and peak area is that 63158, RSD is 5.08%, accounts for 1.60% of total peak area.
No. 13 peaks, average retention time RT is 48.405min, and RSD is 0.16%, and peak area is that 144521, RSD is 3.92%, accounts for 3.65% of total peak area.
No. 14 peaks, average retention time RT is 50.001min, and RSD is 0.15%, and peak area is that 86223, RSD is 5.29%, accounts for 2.18% of total peak area.
No. 15 peaks, average retention time RT is 59.416min, and RSD is 0.13%, and peak area is that 959208, RSD is 5.85%, accounts for 24.24% of total peak area.
As mentioned above, contain altogether the total peak of 15 features in the finger-print of four refreshing sheets, wherein highest peak is No. 4 peaks, being decided to be with reference to peak is S peak, with reference to peak, is psoralen, and collection of illustrative plates total length is 70min, wherein the peak of the super total peak area 1% of unimodal area has 15, and they are 1～No. 15 peaks; Wherein the peak of the super total peak area 5% of unimodal area has 4, and they are No. 2 peaks, No. 4 peaks, No. 5 peaks, No. 15 peaks; The peak of the super total peak area 10% of unimodal area has 3, is No. 4 peaks, No. 5 peaks, No. 15 peaks.Take No. 4 peaks as with reference to peak, the relative retention time at each peak and relative peak area in Table 2, table 3.
Similarity evaluation: the similarity evaluation software (2004A version) that adopts the Chinese Pharmacopoeia council to recommend carries out comprehensive evaluation to HPLC collection of illustrative plates, similarity evaluation the results are shown in Table 4, table 5, and accompanying drawing 4 is shown in by the collection of illustrative plates after coupling.
The relative retention time at each total peak in 10 batch of four refreshing sheet finger-print of table 2.
| ? | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | A | RSD(%) |
| 1 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.13 |
| 2 | 0.38 | 0.38 | 0.38 | 0.38 | 0.38 | 0.36 | 0.35 | 0.34 | 0.36 | 0.35 | 0.37 | 4.28 |
| 3 | 0.40 | 0.40 | 0.39 | 0.39 | 0.39 | 0.37 | 0.36 | 0.36 | 0.37 | 0.37 | 0.38 | 3.99 |
| 4 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 |
| 5 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 1.03 | 0.00 |
| 6 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 1.27 | 0.04 |
| 7 | 1.29 | 1.29 | 1.29 | 1.28 | 1.28 | 1.28 | 1.28 | 1.28 | 1.28 | 1.28 | 1.28 | 0.03 |
| 8 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 1.31 | 0.04 |
| 9 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 0.06 |
| 10 | 1.46 | 1.46 | 1.46 | 1.45 | 1.45 | 1.45 | 1.45 | 1.45 | 1.45 | 1.45 | 1.45 | 0.05 |
| 11 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 1.53 | 0.05 |
| 12 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 1.55 | 0.06 |
| 13 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 1.60 | 0.06 |
| 14 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 1.65 | 0.07 |
| 15 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 1.96 | 0.09 |
The relative peak area at each total peak in table 3.10 batch four refreshing sheet finger-prints
| ? | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | A | RSD(%) |
| 1 | 0.12 | 0.16 | 0.13 | 0.13 | 0.13 | 0.17 | 0.15 | 0.17 | 0.15 | 0.14 | 0.14 | 12.58 |
| 2 | 0.29 | 0.27 | 0.27 | 0.27 | 0.26 | 0.26 | 0.26 | 0.24 | 0.22 | 0.21 | 0.26 | 8.81 |
| 3 | 0.13 | 0.15 | 0.14 | 0.15 | 0.15 | 0.14 | 0.13 | 0.16 | 0.16 | 0.17 | 0.15 | 9.04 |
| 4 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 |
| 5 | 0.85 | 0.85 | 0.85 | 0.85 | 0.85 | 0.85 | 0.85 | 0.85 | 0.84 | 0.84 | 0.85 | 0.61 |
| 6 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.15 | 0.14 | 0.55 |
| 7 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.60 |
| 8 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.51 |
| 9 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 1.41 |
| 10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.43 |
| 11 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 1.05 |
| 12 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 1.01 |
| 13 | 0.16 | 0.15 | 0.16 | 0.17 | 0.15 | 0.16 | 0.16 | 0.16 | 0.17 | 0.17 | 0.16 | 3.24 |
| 14 | 0.10 | 0.10 | 0.10 | 0.10 | 0.09 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 1.33 |
| 15 | 1.07 | 1.07 | 1.07 | 1.07 | 1.07 | 1.07 | 1.07 | 1.07 | 1.08 | 1.07 | 1.07 | 0.29 |
Table 4.10 batch four refreshing sheet sample similarity evaluation results
| ? | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Reference fingerprint |
| S1 | 1.000 | 0.986 | 0.983 | 0.990 | 0.989 | 0.972 | 0.965 | 0.964 | 0.959 | 0.988 | 0.991 |
| S2 | 0.986 | 1.000 | 0.976 | 0.984 | 0.990 | 0.975 | 0.960 | 0.969 | 0.967 | 0.985 | 0.990 |
| S3 | 0.983 | 0.976 | 1.000 | 0.986 | 0.981 | 0.959 | 0.946 | 0.956 | 0.965 | 0.977 | 0.985 |
| S4 | 0.990 | 0.984 | 0.986 | 1.000 | 0.996 | 0.964 | 0.949 | 0.965 | 0.955 | 0.981 | 0.988 |
| S5 | 0.989 | 0.990 | 0.981 | 0.996 | 1.000 | 0.975 | 0.960 | 0.976 | 0.965 | 0.987 | 0.993 |
| S6 | 0.972 | 0.975 | 0.959 | 0.964 | 0.975 | 1.000 | 0.988 | 0.988 | 0.983 | 0.984 | 0.990 |
| S7 | 0.965 | 0.960 | 0.946 | 0.949 | 0.960 | 0.988 | 1.000 | 0.984 | 0.980 | 0.974 | 0.982 |
| S8 | 0.964 | 0.969 | 0.956 | 0.965 | 0.976 | 0.988 | 0.984 | 1.000 | 0.989 | 0.973 | 0.988 |
| S9 | 0.959 | 0.967 | 0.965 | 0.955 | 0.965 | 0.983 | 0.980 | 0.989 | 1.000 | 0.969 | 0.985 |
| S10 | 0.988 | 0.985 | 0.977 | 0.981 | 0.987 | 0.984 | 0.974 | 0.973 | 0.969 | 1.000 | 0.993 |
| DZ | 0.991 | 0.990 | 0.985 | 0.988 | 0.993 | 0.990 | 0.982 | 0.988 | 0.985 | 0.993 | 1.000 |
Table 5.10 batch sample similarity evaluation result 2
| Lot number | Similarity (reference) | Similarity (contrast) |
| S1 | 1.000 | 0.991 |
| S2 | 0.986 | 0.990 |
| S3 | 0.983 | 0.985 |
| S4 | 0.990 | 0.988 |
| S5 | 0.989 | 0.993 |
| S6 | 0.973 | 0.990 |
| S7 | 0.965 | 0.982 |
| S8 | 0.964 | 0.988 |
| S9 | 0.959 | 0.982 |
| S10 | 0.988 | 0.993 |
embodiment 3four refreshing sheet standard finger-prints and each total peak ownership
The generation of 3.1 standard finger-prints
Get 10 batches of four refreshing sheets, by embodiment 1 condition, detect, obtain the HPLC collection of illustrative plates of 10 batch samples, as shown in Figure 5.Adopt similarity evaluation software (2004A version) to carry out similarity evaluation, method is with embodiment 2, and generates standard finger-print, sees Fig. 5.
The ownership at 3.2 each total peaks of finger-print
Measure the finger-print of each medicinal material in four refreshing sheets in accordance with the law, and it is contrasted respectively to collection of illustrative plates importing fingerprint similarity evaluation software (similarity evaluation with four refreshing sheets, 2004A version) in, by comparing chromatographic peak retention time, represent its contribution to total peak, determine the ownership of four refreshing sheet sample finger-prints on medicinal material collection of illustrative plates.
3.2.1 nutmeg medicinal materials fingerprint
Nutmeg medicinal materials fingerprint chromatogram is shown in Fig. 6, and total peak ownership is in Table 6.
The total peak of the refreshing sheet sample of table 6. four ownership nutmeg medicinal material
| Total peak number | Retention time mean value | Retention time RSD% |
| 9 | 42.359 | 0.10 |
| 10 | 44.085 | 0.19 |
| 11 | 46.327 | 0.19 |
3.2.2 evodia rutaecarpa medicinal materials fingerprint
Evodia rutaecarpa medicinal materials fingerprint chromatogram is shown in Fig. 7, and total peak ownership is in Table 7.
The total peak of the refreshing sheet sample of table 7. four ownership evodia rutaecarpa medicinal material
| Total peak number | Retention time mean value | Retention time RSD% |
| 8 | 39.612 | 0.25 |
3.2.3 corylifolia L finger-print
Corylifolia L finger-print chromatogram is shown in Fig. 8, and total peak ownership is in Table 8.
The total peak of the refreshing sheet sample of table 8. four ownership corylifolia L
| Total peak number | Retention time mean value | Retention time RSD% |
| 4 | 30.305 | 0.29 |
| 5 | 31.075 | 0.30 |
| 6 | 38.518 | 0.25 |
| 12 | 46.884 | 0.22 |
| 13 | 48.405 | 0.23 |
| 14 | 50.001 | 0.23 |
| 15 | 59.416 | 0.10 |
3.2.4 schisandra chinensis medicinal material finger-print
Schisandra chinensis medicinal material finger-print chromatogram is shown in Fig. 9, and total peak ownership is in Table 9.
The total peak of the refreshing sheet sample of table 9. four ownership schisandra chinensis medicinal material
| Total peak number | Retention time mean value | Retention time RSD% |
| 1 | 3.210 | 0.15 |
| 7 | 38.934 | 0.12 |
3.2.5 date medicinal materials fingerprint
Date medicinal materials fingerprint chromatogram is shown in Figure 10, and total peak ownership is in Table 10.
The total peak of the refreshing sheet sample of table 10. four ownership date medicinal material
| Total peak number | Retention time mean value | Retention time RSD% |
| 2 | 11.090 | 0.22 |
| 3 | 11.553 | 0.22 |
In sum, in four refreshing tablet preparation standard diagrams, 15 total peaks all can find clearly ownership, and in prescription, the characteristic peak of the 5 taste medicinal materials such as nutmeg, evodia rutaecarpa, Psoralea corylifolia, the fruit of Chinese magnoliavine, date all can embody in four refreshing tablet preparation finger-prints; In side, ginger medicinal material substantially can't check characteristic peak under this chromatographic condition.
By HPLC chromatogram and the standard finger-print of four refreshing tablet preparations are compared, its similarity is evaluated, can differentiate quickly and efficiently that the true and false of product is good and bad.
Claims (5)
1. a method for building up for four refreshing tablet preparation finger-prints, adopts liquid phase chromatography, it is characterized in that: comprise the following steps:
(1) preparation of reference substance solution: it is appropriate that precision takes psoralen, Isopsoralen, schizandrin reference substance respectively, is mixed with the solution of 10～100 μ g/mL, in contrast product solution with methyl alcohol;
(2) preparation of need testing solution: after four refreshing sheets remove dressing, precision takes 0.1～10g, with the ultrasonic extraction of methyl alcohol, extract filters with miillpore filter, obtains need testing solution;
(3) adopt liquid chromatography for measuring finger-print: the accurate need testing solution of drawing injects high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains finger-print;
(4) adopt the chromatographic condition of liquid chromatography for measuring finger-print: chromatographic column be take octadecylsilane chemically bonded silica as filler; The gradient eluent that mobile phase is comprised of mobile phase A and Mobile phase B, mobile phase A is acetonitrile, Mobile phase B is water; Detect wavelength 220nm～360nm; 20～50 ℃ of column temperatures; Flow velocity 0.5～1.5mL/min; Time 30～90min.
2. the method for building up of four refreshing tablet preparation finger-prints as claimed in claim 1, is characterized in that: the preparation of described step (2) need testing solution is: after four refreshing sheets remove dressing, precision takes 0.1～10g, puts in tool plug conical flask, precision adds methyl alcohol 10～100ml, close plug, weighed weight, soak, after ultrasonic processing, let cool, weighed weight again, supplies the weight of less loss with methyl alcohol, shake up, filter, with miillpore filter, filter, get subsequent filtrate, obtain need testing solution;
Described step (3) adopts liquid chromatography for measuring finger-print to be: accurate need testing solution 5～25 μ L that draw inject high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtain finger-print.
3. the method for building up of four refreshing tablet preparation finger-prints as claimed in claim 1, is characterized in that:
The preparation of described step (2) need testing solution is: after four refreshing sheets are removed dressing, precision takes 0.5g, puts in tool plug conical flask, and precision adds methyl alcohol 25ml, close plug, weighed weight, soaks, ultrasonic processing 45 minutes, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate, obtain need testing solution;
Described step (4) chromatographic condition is: chromatographic column be take octadecylsilane chemically bonded silica as filler; Adopt gradient elution, mobile phase is the gradient eluent that water and acetonitrile form, water (%) 95～20: acetonitrile (%) 5～80; Column temperature: 35 ℃; Detect wavelength 246nm; Flow velocity: 1.0ml/min; Analysis time: 70min.
4. the method for building up of four refreshing tablet preparation finger-prints as claimed in claim 1, is characterized in that: the gradient elution described in step (4), and gradient elution carries out with following volumetric concentration configuration:
In the time of 0 minute, mobile phase A is 5%, and Mobile phase B is 95%;
In the time of 20 minutes, mobile phase A is 30%, and Mobile phase B is 70%;
In the time of 25 minutes, mobile phase A is 40%, and Mobile phase B is 60%;
In the time of 60 minutes, mobile phase A is 80%, and Mobile phase B is 20%;
In the time of 70 minutes, mobile phase A is 80%, and Mobile phase B is 20%.
5. the method for building up of four refreshing tablet preparation finger-prints as claimed in claim 1, is characterized in that the four refreshing tablet preparation HPLC finger-prints that make have 15 total peaks, and peak number is 1～15, take wherein characteristic peak (4) psoralen peak as with reference to peak, peak number; Relative retention time is respectively: 1(0.11), 2(0.37), 3(0.38), 4(1.00), 5(1.03), 6(1.27), 7(1.28), 8(1.31), 9(1.40), 10(1.45), 11(1.53), 12(1.55), 13(1.60), 14(1.65), 15(1.96).
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| CN107884505A (en) * | 2017-11-13 | 2018-04-06 | 上海和黄药业有限公司 | A kind of detection method of antideaf otic pill finger-print and its application |
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Application publication date: 20141210 |